Highly sensitive chemiluminescent method for the detection of cell contamination.

Minisatellite analysis is commonly used in forensic disputes but can also be applied to the investigation of cell contamination. Such a problem arises, for example, when transplantation is performed. The presence of contamination has been investigated by other authors using radioactive methods. In the present study we describe a method that allows the detection of contamination with high sensitivity without using radioactive substances. Our technique is based on the use of polymerase chain reaction (PCR) amplification of minisatellite sequences (VNTR), followed by chemiluminescent detection. In particular, biotin-labelled dCTP is included in the PCR mixture and detection of PCR products is obtained following the CSPD chemiluminescent protocol (Southern-Light Nucleic Acid Detection Systems). We applied this method to artificial mixes of DNA of two individuals with alleles of different sizes. We performed progressive dilutions of an individual DNA into the other's DNA and revealed a contamination of 1 in 2500 cells. We also tested our technique searching for maternal contamination in cord blood samples in 60 cases and revealed a 18.3% contamination. The technique that we set up proves to be a very sensitive one which could be applied not only to the detection of maternal cells in cord blood but also in studying any other kind of contamination.

A retrospective evaluation of HLA-A, B and -DRB1 matching in liver transplantation.

Studies on the influence of histocompatibility in liver transplantation have not produced clearcut results. We retrospectively studied the influence of HLA-A, B and -DRB1 matching on the survival of 517 liver-transplanted patients using univariate analysis. The following parameters were also considered in relation to transplant outcome: donor and recipient age, original disease, transplant center, and pretransplant blood transfusions. Twenty-four-month graft survival according to the number of HLA-A, B, DRB1 mismatches (MM) was 70.9% (n = 28) for zero to two MM, 76.6% (n = 248) for three to four MM, and 73.1% (n = 241) for five to six MM (P = 0.7). We obtained similar results when considering HLA-A, B MM alone. Survival rates according to HLA-DRB1 MM were 71.7% (n = 36) for zero MM, 73.7% (n = 236) for one MM, and 76.4% (n = 245) for two MM (P = 0.6). The same analyses, performed on cirrhotic patients alone, gave identical results. In conclusion, this study suggests, on a large series of patients, that HLA compatibility has no influence on liver transplant survival. On the contrary, an influence on transplant outcome was found for donor age, transplant center, and original disease.

Detection of maternal DNA in human cord blood stored for allotransplantation by a highly sensitive chemiluminescent method.

Human cord blood (CB), a rich source of hematopoietic stem and progenitor cells, is currently used for bone marrow reconstitution. However, the level of contamination of CB with maternal cells that could provoke graft-versus-host disease (GvHD) is a matter of concern. In the present study, 60 consecutive CB samples collected and stored in the Milan CB Bank, for which no maternal DNA was detected through genomic HLA typing, were examined to ascertain maternal cell contamination using polymerase chain reaction amplification of two minisatellites, apolipoprotein B gene (ApoB) and D1S80, followed by chemiluminescent detection. The sensitivity of the method employed in this study was 0.04%, comparable to that of radioactive methods. A maternal specific allele was found in 11 of the 60 CB units, at a level ranging from 1:100 to 1:2500. We could also detect the child paternal allele in 3 of the 30 mothers whose newborn was heterozygous at the loci examined. Our study indicates that maternal cells are present in 18.3% of the 60 samples examined. The clinical relevance of such a presence remains to be established. In our opinion, information on maternal cell contamination should be included within the quality control tests performed before delivering a unit.